Original Article Development of Simple Protocol For

International Journal of Medical Laboratory 2019;6(1):51-62.

Original Article

Development of Simple Protocol for Generation of Functionally Active Hepatocyte-like Cells from Human Adipose Tissue-derived Stem Cells

Sahere Rouzbehan1,2 M.Sc., Nahid Davoodian2,3* Ph.D., Ali Jamshidi4 M.D., Ali Atashabparvar5 M.D., Najmeh Davoodian6 Ph.D.

1Endocrinology and Metabolism Research Center, Hormozgan University of Medical Sciences, Bandar Abbas, Iran. 2Department of Clinical Biochemistry, Faculty of Medicine, Hormozgan University of Medical Sciences, Bandar Abbas, Iran. 3Molecular Medicine Research Center, Hormozgan Health Institute, Hormozgan University of Medical Sciences, Bandar Abbas, Iran. 4Department of Surgery, Faculty of Medicine, Hormozgan University of Medical Sciences, Bandar Abbas, Iran. 5Department of Pathology, Faculty of Medicine, Hormozgan University of Medical Sciences, Bandar Abbas, Iran. 6Research Institute of Animal Embryo Technology, Shahrekord University, Shahrekord, Iran. A B S T R A C T

Article history Background and Aims: Human adipose tissue-derived stem cells (hASCs) are Received 18 Dec 2018 considered as an attractive source of regenerative stem cells, mainly because of Accepted 14 Jan 2019 their higher proliferation rate, more accessibility and hepatocyte like properties Available online 10 Mar 2019 as compared to mesenchymal stem cells isolated from other tissues. Numerous Key words studies have described the beneficial use of adipose tissue-derived stem cells for Fibroblast growth factor generating hepatocyte-like cells. However, due to the lack of appropriate culture Hepatic differentiation conditions, most of the produced cells exhibit poor functionality. The aim of the Hepatocyte-like cells Mesenchymal stem cells present study was to establish a new protocol for the efficient hepatic differentiation of hASCs. Materials and Methods: hASCs were cultured in hepatic differentiation medium containing fibroblast growth factor 4, hepatocyte growth factor, dexamethasone and oncostatin M using a three-step protocol up to 21 days. Then, the functionality of the treated cells was evaluated by analyzing specific hepatocyte genes and biochemical markers at various time points. Results: A significant upregulation in albumin, alpha-fetoprotein, cytokeratin 18 and hepatocyte nuclear factor-4α expressions was observed in differentiated cells relative to day 1 of differentiation protocol. Moreover, the finding of glycogen deposits increased urea production and positive immunofluorescence staining for albumin and alpha-fetoprotein in hepatocyte-like cells suggesting that most of the cells differentiate into hepatocyte-like cells. Conclusions: Our report has provided a simple protocol for differentiation of hASCs into more functional hepatocyte-like cells.

*Corresponding Author: Department of Clinical Biochemistry, Faculty of Medicine, Hormozgan University of Medical Sciences, Bandar Abbas, Iran. Tel: +987633710373, Fax: +987633668478, Email: [email protected] S. Rouzbehan et al.

Introduction

The liver is a significant organ in the human hepatocyte like properties [10] as compared to body performing vital functions including MSCs isolated from other tissues. detoxification, metabolism, and hormone There are an increasing number of studies balance. In addition to these roles, liver is offering ASCs as an attractive candidate known to possess a remarkable regenerative for generating hepatocyte-like cells. These capacity. However, this ability is impaired studies have provided support for the use following numerous liver failures such as of a combination of growth factors and cirrhosis for which liver transplantation is the inductive agents to induce ASCs toward only available clinical option [1]. Indeed, hepatic lineage [11-15]. However, most of due to discrepancies associated with liver the produced hepatocyte-like cells exhibit transplantation, it is of utmost importance to poor functionality, mainly due to the find alternative strategies for the treatment of lack of appropriate induction conditions. end-stage liver disorders [2]. Developmental studies have uncovered a number In this context, cell-based therapy has become of transcription factors and regulators as being the focus of intense investigation in recent implicated in hepatocyte differentiation [16]. years [3]. Therefore, various kinds of stem In this regard, fibroblast growth factors (FGFs) cells have been studied to examine their have been identified as being involved in clinical applications in regenerative medicine early development and organogenesis [17, [4]. To date, there are numerous studies 18]. Of particular importance is FGF4 describing the beneficial use of mesenchymal because of its involvement in hepatic stem cells (MSCs) for liver cell therapy, induction [16, 19] as well as its role in liver mainly because of their multipotent potential, regeneration [20]. In addition, it has been lack of ethical concerns, and risk of rejection reported that MSCs pretreated with FGF4 in [5, 6]. MSCs can be isolated from different combination with hepatocyte growth factor kinds of tissues including bone marrow (the (HGF) is able to reduce liver fibrosis and first known tissue as a source of MSCs) [7], improve liver function in CCl4 induced mice umbilical cord blood, amniotic fluid as well as [21]. Similar results have also been described adipose tissue [8]. Of particular interest to upon using of FGF4 transduced bone marrow researchers are the human adipose tissue- MSCs (BM-MSCs) in the cirrhotic liver derived stem cells (hASCs) which for the first model [22]. time were isolated and described by Zuk et al. Recently, using a combination of inductive In addition to their multilineage potential [9], agents including HGF, oncostatin M, and hASCs have been reported to exhibit higher dexamethasone, we have differentiated hASCs proliferation rate, more accessibility and into hepatocyte-like cells [23]. Based on the mentioned results vividly showing

International Journal of Medical Laboratory 2019;6(1): 51-62. 52 PRODUCTION OF HEPATOCYTE-LIKE CELLS the important role of FGF4 in hepatic after hepatic induction. Three independent differentiation, the current study was designed experiments were conducted in this study. to examine a new protocol for generation RNA extraction and quantitative real time of functionally active hepatocyte-like cells polymerase chain reaction (qRT-PCR) from hASCs. analysis Total RNA was extracted using TRI reagent Materials and Methods (Sigma-Aldrich, USA) on days 1, 7, 14, Isolation and culture of hASCs and 21 of differentiation protocol. After hASCs were isolated from human lipoaspirates DNase (Fermentas, USA) treatment, Reverse as described previously [23]. Briefly, transcription and cDNA amplification was lipoaspirates were digested using 0.075% performed with cDNA synthesis kit (Thermo collagenase I (Sigma-Aldrich, USA) at 37ºC Ficher, USA) and SYBR Premix Ex Taq II for 30 min. Finally, the extracted cells were (Takara, Japan) respectively. Using specific suspended in Dulbecco’s modified Eagle’s primers (Table 1), qRT-PCR was conducted medium (DMEM) (Gibco, USA) containing with a three-step procedure as follows: 10% fetal bovine serum (FBS) (Gibco, USA), denaturation at 95°C for 5 s, annealing at 60°C and 1% penicillin/streptomycin (Sigma- for 30 s and extension at 72°C for 30 s, for a Aldrich, USA) and incubated in a humidified total of 40 cycles. As a reference gene, β-actin atmosphere at 37º C with 5% CO2. expression was used for normalization and In vitro hepatic differentiation of hASCs RNA levels were evaluated in triplicate. To induce hepatic differentiation of hADSCs, Finally, 2-ΔΔCt method was applied to calculate a three-step procedure was used (Fig. 1A]. At relative quantification of gene expression. passage 3, the cells (1×103 cells/cm2) were Immunofluorescence Staining cultured in DMEM supplemented with 10 % After fixation with 4% paraformaldehyde FBS, 20 ng/ml HGF (Biolegend, USA), 10-7 solution for 10 min at room temperature, the mol/l dexamethasone (Sigma-Aldrich, USA) cells were permeabilized using 0.2% Triton X- together with 10 ng/ml FGF4 (Biolegend, 100 for 30 min. A solution containing 1% USA]. After 3 days, the cells were treated with bovine serum albumin (BSA) and 0.1% Triton the inductive agents as step one in the absence X-100 in PBS was used for blocking, followed of FGF4, followed by the addition of by washing the cells with phosphate buffer oncostatin M (Biolegend, USA) to the medium solution (PBS) and incubation with 1:200 at concentration of 10 ng/ml for the next 2 monoclonal anti-human albumin (Abcam, weeks. The culture medium was renewed USA) and 1:200 monoclonal anti-human alpha every 3 days and hepatic differentiation assays 1 fetoprotein (Abcam, USA) overnight at 4ºC. were carried out at 1, 7, 14 and 21 days

53 International Journal of Medical Laboratory 2019;6(1):51-62. S. Rouzbehan et al.

Table 1. Primer sequences for qRT-PCR analysis.

Genes Primer Sequences Sense 5'-GAGACCAGAGGTTGATGTGATG -3' Albumin Antisense 5'-AGGCAGGCAGCTTTATCAGCA-3' Sense 5'-CATGAGCACTGTTGCAGAGGAGA -3' Alpha-fetoprotein Antisense 5'-CGTGGTCAGTTTGCAGCATTCTG -3' Sense 5'-TTGATGACACCAATATCACACGA- 3' Cytokeratin 18 Antisense 5'-TATTGGGCCCGGATGTCTG-3' Sense 5'-GCGGCCAACGGCGAGCTA -3' Cytokeratin 19 Antisense 5'-GCAGGACAATCCTGGAGTTCTC -3' Sense 5'-CTTCTTTGACCCAGATGCCAAG-3' Hepatocyte nuclear factor-4α Antisense 5'-GAGTCATACTGGCGGTCGTTG-3' Sense 5'-CTGGAACGGTGAAGGTGACA-3' β-actin Antisense 5'-AAGGGACTTCCTGTAACAATGCA -3'

The next day, cells were treated with Alexa acid for 5 min. After washing with PBS, the cells Flour 594 donkey anti-mouse IgG (1:500, were treated with Schiff’s reagent (Sigma– Abcam, USA) for 1 h and counterstained Aldrich, USA) for 15 min. and counterstained with 4,6-diamidino-2-phenylindole to visualize using hematoxylin. Finally, the cells were nuclei. Finally, the cells were examined visualized under an inverted microscope [23]. with an inverted fluorescence microscope Urea production (Labomed TCM 400). At least 3 images form Urea concentrations were measured in the each cover slips, 3 cover slips per each sample culture media of induced ASCs at different and 3 samples (biological replicates, n=3) time points (on days 1, 7, 14, and 21). First, per each treatment were considered for the cells were incubated in a culture medium quantification of data [24]. In addition, all containing 3mM NH4CL for 24h at 37ºC. images were taken with exactly the same After collecting the supernatants, urea acquisition parameters including exposure production was evaluated with a colorimetric time, gamma and saturation. Finally, image assay kit (Zistchem, Iran) following the J software (version 1.42 V, NIH, USA) was manufacturer’s instruction. As a negative used for quantification. control, untreated hASCs were used and the Hepatic functional tests amount of urea was normalized to the number Glycogen staining of cells in each well [23]. This protocol At day 21, the cells were evaluated for detection was approved by Hormozgan University of of glycogen deposit using periodic acid-schiff Medical Sciences Ethics Committee. (PAS) staining. Firstly, the cells were fixed Statistical analysis with 4% paraformaldehyde solution for 10 The data are expressed as mean±SD. One way- min, followed by oxidation using 1% periodic analysis of variance followed by Dunnett post

International Journal of Medical Laboratory 2019;6(1): 51-62. 54 PRODUCTION OF HEPATOCYTE-LIKE CELLS hoc test was conducted for analysis of were evaluated morphologically. During the statistical comparison and the p-value <0.05 first week of induction, the cells exhibited a was considered to be significant. fibroblast-like shape without any significant morphological changes. After 2 weeks, Results however, the induced hASCs slowly lost Morphological changes and expression of fibroblastic morphology and started becoming hepatocyte-specific markers during hepatic broad and flattened. Notably, morphological differentiation of hASCs changes were remarkable during the last To induce differentiation of hASCs into week of differentiation and some of the cells hepatocyte-like cells, medium containing FGF4, exhibited hepatocyte-like morphology such as HGF, dexamethasone, and oncostatin M was a polygonal and round shape and cytoplasmic used over the course of 21 days (Fig.1A). At granulation (Fig. 1B). various stages of differentiation protocol, cells

Fig. 1. Hepatic differentiation of hASCs. A: Schematic representation of the hepatic differentiation protocol. B: Morphology of hASCs treated with HGF= hepatocyte growth factor, Dex= dexamethasone, OSM= oncostatin M, and FGF4= fibroblast growth factor at days 1, 7, 14, and 21. The cells exhibited remarkable changes toward round and polygonal shapes at days 14 and 21 upon hepatic induction.

The differentiation status was also confirmed mentioned genes was measured. As a negative by evaluating the expression of several and positive controls, untreated hASCs and hepatocyte specific genes, including albumin, Hep G2 cells were used, respectively. alpha-fetoprotein, cytokeratin 18, cytokeratin As illustrated in Fig. 2A, an early increase was 19, and hepatocyte nuclear factor-4α to observed for alpha-fetoprotein mRNA, an examine whether the morphological changes early hepatic progenitor marker at day 7 to were associated with the hepatic genes 3-fold (p<0.05) with further increase at days expression. Therefore, total RNA of treated 14 and 21 to 8 (p<0.001) and 9.5 fold cells were extracted at various time points (on (p<0.001) respectively. Moreover, statistical days 1, 7, 14, 21) and the expression of the analysis validated a significant upregulation

55 International Journal of Medical Laboratory 2019;6(1):51-62. S. Rouzbehan et al. for albumin expression level at day 14 to 5.5 was found to significantly upregulate at fold (p<0.01) that reached maximum level of day 21 to approximately 5-fold (p<0.01) in 13-fold (p<0.001) at day 21 compared to day 1 comparison to day 1 (Fig. 2C). Significant of hepatic differentiation induction (Fig. 2B). expression level of hepatocyte nuclear However, upon hepatic differentiation of factor α was noted starting at day 14 (3.5- hASCs, no significant difference in cytokeratin fold, p<0.05) with a maximum increase at 19 expression level was detected at all-time day 21 to nearly 8-fold upon hepatic points (Fig. 2D). By contrast, the mRNA level induction of hASCs (Fig. 2E). of cytokeratin 18, as an adult hepatic marker,

Fig. 2. qRT-PCR analysis of several hepatocyte-specific genes upon the treatment of hASCs with a combination of hepatocyte growth factor (HGF), dexamethasone (DEX), oncostatin M (OSM), and fibroblast growth factor (FGF4). The expression levels of target genes were normalized with β-actin. The data are shown as mean±SD in three independent experiments (n=3). ALB= Albumin; AFP= Alpha-fetoprotein; CK= Cytokeratin. *p<0.05, **p<0.01, ***p<0.001 relative to day 1.

International Journal of Medical Laboratory 2019;6(1): 51-62. 56 PRODUCTION OF HEPATOCYTE-LIKE CELLS

In addition, the expression of albumin the high percentage of alpha-fetoprotein (Fig. and alpha-fetoprotein genes were further 3B) and albumin (Fig. 4B) positive cells in confirmed at protein level. Consistent with hepatocyte-like cells. By contrast, untreated the expression of mentioned genes, the hADSCs were negative whereas the staining treated cells stained positive for both alpha- was intensely positive for HepG2 cells fetoprotein (Fig. 3A) and albumin (Fig. 4A) at (Figs. 3, 4). day 21. Also, quantification of data revealed

Fig. 3. Immunofluorescence staining of alpha-fetoprotein (AFP). A: More intensive staining was detected for AFP in hepatocyte-like cells compared to the untreated cells. B: Quantification of immunostaining results provided in A. ***p<0.001 relative to untreated hASCs, (n=3).

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Fig. 4. Immunofluorescence staining of albumin. A: More intensive staining was detected for albumin in hepatocyte-like cells compared to the untreated cells. B: Quantification of immunostaining results provided in A. ***p<0.001 relative to untreated hASCs, n=3.

Functional characterization of hepatocyte- strongly positive staining in the treated cells like cells derived from hASCs at day 21. However, no deposit of glycogen To verify the biological functions of treated was found in non-induced cells, while Hep cells, urea production and glycogen storage G2 cells were intensely positive for PAS ability of the cells were examined. For this staining (Fig. 5A). reason, PAS was applied to assess the In addition to these results, metabolic function glycogen deposit in the cells. As expected, of hepatocyte-like cells was also evaluated hepatic differentiation of hASCs led to using urea assay at various stages of

International Journal of Medical Laboratory 2019;6(1): 51-62. 58 PRODUCTION OF HEPATOCYTE-LIKE CELLS hepatic differentiation. Following 2 weeks of and continued to increase gradually with a hepatic induction, urea secretion elevated to maximum level to 17 mg/dl/50000 cells approximately 15 mg/dl/50000 cells (p<0.05) (p<0.01) compared to day 1 (Fig. 5B).

Fig. 5. Functional assays of hepatocyte-like cells. A: Glycogen storage ability was detected using periodic acid-schiff staining which showed more positive cells in hepatocyte-like cells compared to untreated hASCs. B: A significant increase in urea production was detected in hepatocyte-like cells at day 14 and day 21 relative to day 1. The data are shown as mean±SD in three independent experiments (n=3). *p <0.05, **p <0.01, ***p <0.001 relative to day 1.

Discussion

As it has been extensively reported, ASCs level than a real hepatocyte mainly due to possess the potential to differentiate into using inefficient induction conditions. We hepatocyte-like cells using a cocktail of have recently documented the hepatic growth factors and inductive agents [11-13, differentiation of hASCS using a combination 15]. Interestingly, the expression of several of HGF, dexamethasone, and oncostatin M hepatocyte-specific markers in naïve hASCs over the course of 21 days [23]. In the current shows them to become one of the most study, we report the differentiation efficacy of attractive sources for cell-based therapy of new culture medium by adding FGF4 to liver diseases [10]. Whereas these findings are previous inductive medium to promote exciting, the function of the generated differentiation of hASCs toward functional hepatocyte-like cells is often at a far lower hepatocyte-like cells.

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FGFs, secreted from the cardiac mesoderm, significant increase in alpha-fetoprotein, are known to be involved in early liver albumin, cytokeratin 18, and hepatocyte development [18]. In this regard, it has been nuclear factor-4α expression levels was well documented that FGFs are required for detcted in a time-dependent manner. Our albumin expression and eventually hepatic study also revealed that new differentiation induction at early somatic stages of condition induces hASCs toward functional development [25]. Of particular interest is hepatocyte-like cells as shown by the presence FGF4, a member of FGF superfamily, acting of glycogen storage and increase in urea as a mitogenic, angiogenic, and survival factor production as being two prominent features of and involving in cell proliferation and hepatocyte. These findings indicate that using differentiation [26]. Notably, the striking role a cocktail of the mentioned inductive agents of FGF4 in enrichment and propagation of results in differentiation of hASCs into hepatic progenitors has been reported [27]. functional hepatocyte-like cells. Added to this, improvement in liver In this context, it is important to mention regeneration has been demonstrated upon that growth factors and regulators used in transplantation of FGF4 transduced BM-MSCs this study are known to be involved in in cirrhotic rats [22]. In support of this, FGF4 development and differentiation of hepatocyte. together with HGF were found to promote In addition to acting as a hepatocyte mitogen, hepatic differentiation of MSCs and these HGF is also required for hepatoblast migration pretreated cells have been shown as having the and proliferation [28, 29]. As mentioned capacity to improve liver function in CCl4- before, FGFs exert a major influence on induced liver fibrosis [21]. hepatic specification and also promote liver Based on the mentioned results clearly bud growth [25, 27]. Moreover, it is well confirming the important role of FGF4 in liver established that oncostatin M, secreted by development and regeneration, in this study haematopoietic cells in liver, promotes we examined a new protocol to induce hASCs hepatocyte differentiation, acting in toward functional hepatocyte-like cells. For collaboration of HGF and glucocorticoid this reason, a three-step protocol was designed hormones [30]. in which hASCs were treated with a Conclusion combination of HGF, dexamethasone, FGF4, and oncostatin M, over a course of 21 days and A number of in vitro studies have reported the thereafter differentiation status was evaluated generation of hepatocyte-like cells using a at various time points. Upon using this new combination of growth factors and regulators, condition, treated cells exhibited remarkable including HGF, EGF, FGF, oncostatin M, morphological changes. While we did not dexamethasone, etc. However, given the observe any change in the cytokeratin 19 complexity of liver development, the produced expression level (a cholangiocytes gene), a hepatocyte-like cells exhibit only a certain

International Journal of Medical Laboratory 2019;6(1): 51-62. 60 PRODUCTION OF HEPATOCYTE-LIKE CELLS level of differentiation mainly due to establishing efficient protocols, it is possible inefficient culture conditions. In the current to generate mature functional hepatocyte study, we report the differentiation efficacy from stem cells. of a new, simple and cost-effective culture Conflict of Interest medium containing FGF4, HGF, dexametha- The authors declare no conflict of interest to sone, and oncostatin M to induce hepatic disclose. differentiation of hASCs toward functional Acknowledgements This research was supported in part by a grant hepatocyte-like cells. However, the [75860] from Council for Stem Cell Sciences involvement of additional growth factors in and Technologies as well as by a grant [9433] from Research Vice-Chancellor of Hormozgan hepatocyte differentiation remains to be University for Medical Science (HUMS). studied. Certainly, given the advances in

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